USPTO Patents Application 09864891

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(19) Wo rid Intellectual Property Organization
International Bureau

(43) International Publication Date
25 October 2001 (25.10.2001)

PCT

(10) International Publication Number

WO 01/78591 Al

(51) International Patent Classification 7 :
B65D 81/00

A61B 5/00, (74) Common Representative: MERCK & CO., INC.; 126
East Lincoln Avenue, Rahway, NJ 07065-0907 (US).

(21) International Application Number: PCT/USOl/1 1662

(22) International Filing Date: 9 April 2001 (09.04.2001)

(25) Filing Language: English

(26) Publication Language: English

(30) Priority Data:

60/196,744

12 April 2000 (12.04.2000) US

(71) Applicant (for all designated States except US): MERCK
& CO., INC. [US/US]; 126 East Lincoln Avenue, Rahway,
NJ 07065-0907 (US).

(72) Inventors; and

(75) Inventors/Applicants (for US only): BLAKE, Kevin, R.
[US/US]; 126 East Lincoln Avenue, Rahway, NJ 07065-
0907 (US). CHENG, Kang [US/US]; 126 East Lincoln Av-
enue, Rahway, NJ 07065-0907 (US). CLARKE, Glenn, A.
[US/US]; 126 East Lincoln Avenue, Rahway, NJ 07065-
0907 (US). KATH, Gary, S. [US/US]; 126 East Lincoln
Avenue, Rahway, NJ 07065-0907 (US). KING, Gregory,
W. [US/USJ; 126 East Lincoln Avenue, Rahway, NJ 07065-
0907 (US). WU, Tsuei-Ju [US/US]; 126 East Lincoln Av-
enue, Rahway, NJ 07065-0907 (US).

(81) Designated States (national): AE, AG, AL, AM, AT, AU,
AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM,
HR, HU, ID, IL, IN, IS, JP, KE, KG, KR, KZ, LC, LK, LR,
LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ,
NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM,
TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW.

(84) Designated States (regional): ARTPO patent (GH, GM,
KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), Eurasian
patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
patent (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE,
IT, LU, MC, NL, PT, SE, TR), OAPI patent (BF, BJ, CF,
CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG).

Published:

— with international search report

— before the expiration of the time limit for amending the
claims and to be republished in the event of receipt of
amendments

For two-letter codes and other abbreviations, refer to the "Guid-
ance Notes on Codes and Abbreviations " appearing at the begin-
ning of each regular issue of the PCT Gazette.

(54) Title: AUTOMATED BLOOD SAMPLING APPARATUS

10

OS
ID
00

(57) Abstract: An apparatus (10) and process for automatically, repetitively sampling blood from conscious animals (12). The
^ apparatus ( 10) can simultaneously extract blood from a number of conscious, calherized animals { 1 2) at programmable time intervals.

WO 01/78591 PCT/US01V 11662

TITLE OF THE INVENTION

AUTOMATED BLOOD SAMPLING APPARATUS

BACKGROUND OF THE INVENTION
5 The removal or extraction of blood from conscious animals has been

utilized to determine the presence of bacteria, hormones, parasites, toxins, as well as
the metabolism of drugs and other substances in the animal. Heretofore, the removal
of blood from animals has been performed manually or on an as necessary basis.
Therein, the cannulized animal is anaesthetized and the sample is collected as

10 required at intermittent time intervals with a syringe.

A system for automatic collection of small samples of blood from
conscious animals has been described (Clark, R.G. et al y "Automated Repetitive
Microsampling Of Blood: Growth Hormone Profiles In Conscious Male Rats," J.
Endocr. (1986) Vol. Ill, pp. 27-35). Therein rats bearing indwelling intravenous

15 catheters were connected via swivels to a solenoid operated, three-way fitting and

tubing to a multi-channel peristaltic pump, valves and a fraction collector. The tubing
was filled with heparinized saline from a reservoir. Blood samples from the rats are
drawn past the three-way fitting using a peristaltic pump. A small portion of the
blood is pushed past the three-way fitting into a collector and the remainder of the

20 sample is returned to the animal. This system, particularly, if the animal is small, fails
to address the problem of a replacement substance for the animal's blood that has
been removed. The reference further fails to suggest a solution for the problem of
contamination of the blood sample with saline solution or excess dilution of sample
therewith.

25 There is now a need for an automated blood sampling system that can

sample blood from a conscious animal and address the problems of replacement of
sampled blood to maintain the animals metabolism. There is a further need to
address the problem of contamination and excess dilution of the blood sample with
heparinized saline solution. The present invention addresses and resolves the

30 problems associated with the automatic blood sampling system described herein
above.

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WO 01/78591 PCT/US01/1 1662

10

SUMMARY OF THE INVENTION

The present invention is directed to an apparatus for automatically,
repetitively sampling blood from a conscious animal, characterized as:

a) means for confining a conscious, catheterized animal;

b) a cannula having first and second ends, said first end connected to the
catherized animal, said cannula being suitable for sampling blood from the
animal;

c) a cannula valve having first and second ends, said first end connected to the
cannula, said cannula valve opening and closing being computer controlled;

d) a cannula T-fitting having first, second and third ends, said first end connected
15 to the second end of the cannula valve;

e) an outlet valve having first and second ends, said first end connected, via
tubing, to the second end of the cannula T-fitting, said outlet valve opening
and closing being computer controlled;

20

f) a dispensing tip having first and second ends, said first end connected to the
second end of the outlet valve, said second end being suitable for discharging
blood into a sample, collection vial;

25 g) means for horizontal and. vertical movement of the dispensing tip, said means
for movement being computer controlled;

h) an air T-fitting having first, second and third ends, said first end connected to
the third end of the cannula T-fitting;

30

i) an air valve having first and second ends, said first end connected to the
second end of the cannula T-fitting, said second end exposed to the check
valve;

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WO 01/78591 PCT/US01/11662

j) a syringe pump suitable for transferring blood and saline through the

apparatus, said pump having first and second ends, said first end connected,
via tubing, to the third end of the air T-fitdng, said pump being computer
controlled;

5

k) a rotary, programmable valve having four ports for receiving and discharging
fluids, wherein the ports open and close by computer control, said first port
being connected, via tubing, to the second end of the syringe pump;

10 1) a heparinized saline source, said saline source connected to the second port of
the programmable valve;

m) a fraction collector, comprising:

15 i) a base having two horizontally, opposed, parallel tracks, and a single track

horizontally positioned above and perpendicular to the horizontally
opposed, parallel tracks,

ii) a temperature controlled rack in slidable contact with the horizontally
20 opposed, parallel tracks,

iii) means for sliding the rack along the tracks, said means being computer
controlled, and

25 iv) a plurality of sample, collection vials removably located in the rack, said

vials being suitable for receiving samples from the dispensing tip; and

n) computer means for accepting timing commands for collecting samples from the
animal in coordination with the opening and closing of valves, sliding of the
30 temperature controlled rack, pumping of the syringe pump, and dispensing

samples into the collection vials,

wherein samples can be collected at predetermined time intervals.

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WO 01/78591 PCTAJS01/11662

Hie invention is also directed to a process for automatically collecting
samples from a conscious animal utilizing the apparatus described herein.

BRIEF DESCRIPTION OF THE DRAWINGS
5 FIG. 1 illustrates a front view in elevation of the automatic blood

sampling apparatus; and

FIG. 2 illustrates a front view in elevation of the automatic blood
sampling apparatus with computer means for controlling a process of sampling blood;

10

FIG. 3 illustrates a front, schematic view in elevation of a blood
sample collected in accordance with the process of the invention; and

FIG. 4 a-n illustrates a series of front views in elevation of a process .
15 for collecting a blood sample.

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein is directed to an apparatus for
automatically, repetitively sampling blood from a conscious animal. The various

20 components of the apparatus, i.e. valves, pump and vacuum means, and fraction
collector, can be computer controlled and programmed to collect samples from the
animal at prescribed times.

Referring to HG. 1, apparatus (10) is characterized as a series of
automated components, i.e. valves, fittings and pumps, connected by flexible or rigid

25 tubes, wherein many of the components being computer controlled. An animal (12) to
be sampled is confined to metabolic cage (14) with ample food and water. After the
animal is anaesthetized, a catheter containing heparinized saline solution is implanted
into a vein thereof. The catheter is then exteriorized via an intravenous cannula (16),
and the cannula connects to swivel (18) to allow free movement of the animal within

30 the cage. The swivel connects, via flexible tubing, to cannula valve (20), wherein the
opening and closing of the valve is computer controlled. The cannula valve connects,
via flexible tubing, to a three-port, cannula T-fitting (22). A second end of the
cannula T-fitting connects, via flexible tubing, to outlet valve (24), wherein the
opening and closing of the valve is computer controlled. The outlet valve connects to

35 rigid, dispensing tip (26). Typically, the outlet valve and the dispensing tip are

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WO 01/78591 PCT/US01/11662

positioned on a computer controlled, base (not shown) for reciprocating, vertical
movement. A third end of the cannula T-fitting connects, via flexible tubing, to three-
way, air T-fitting (28). The air T-fitting connects to air valve (30) and the air valve
connects to an ambient atmosphere inlet, optionally via a check valve. Air T-fitting
5 (28) is further connected, via flexible tubing, to syringe pump (32) that provides
means for transfer of fluids through various tubing and components of the apparatus.
The syringe pump connects to a port of multi-port, programmable valve (34). The
other ports of the programmable valve are connected to saline-containing source (36),
distilled water source (38), and acid wash source (40), respectively. Generally, the

10 cannula and outlet valves can be selected from pinch and check valves, as well as
other suitable valves known in the art.

Optionally, depending upon the number of animals to be sampled,
optionally, a plurality of blood sampling lines can be incorporated into the apparatus.
Referring to FIG. 1, a plurality of blood sampling lines characterized as components

15 (14) through (28), components (52) through (56), and associated tubing, can be
connected via syringe pump (32) to syringe pump manifold (42). Syringe pump
manifold (42) connects the plurality of syringe pumps to programmable valve (34).
The programmable valve facilitate common filling and flushing of the lines with
saline solution, acid solution, and distilled water.

20 An additional feature of the invention is a vacuum system to assist in

flushing the apparatus components and tubing of unwanted fluids. Referring to FIG.
1, the vacuum system is illustrated as dispensing tip manifold (50) connected, via
flexible tubing, to waste trap (52). Waste trap (52) connects, via flexible tubing, to
safety trap (54) that connects, via flexible tubing, to vacuum means (56). Dependent

25 upon the number of blood sampling lines incorporated into the apparatus, the

vertically reciprocating base containing the dispensing tips and outlet valves can be
fitted with a dispensing tip manifold to facilitate the removal of waste fluids from the
apparatus. For a plurality of dispensing tips, the horizontal base of the manifold will
contain a plurality of vertical holes therethrough, wherein each tube is adapted to

30 vertical movement through the hole. A vertical hole, intersecting each of the

horizontal holes connects to a vacuum means through a waste trap. During removal
of waste fluids from the apparatus, via the dispensing tip, the discharge end of the
tubes are moved proximal to the vertical holes in the manifold, the vacuum means
applied, and the waste fluids are drawn through the horizontal hole intersecting each

35 vertical hole into the waste trap. The tubing connections to the waste trap should be

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WO 01/78591 PCI7US0 1/1 1662

maintained in a space above the waste fluids to prevent fluid take-up into the vacuum
means. During operation of the blood sampling apparatus, after the dispensing tip
moves vertically, proximal to the first hole of the dispensing tip manifold and the
vacuum means activated, any waste fluids in the tubing can be evacuated therefrom
5 into the waste collection trap. The valve is closed and vacuum means are applied, via
computer controls, and any blood or other liquids within the line between the
dispensing valve and the end of the dispensing tip can be evacuated therefrom by
application of the vacuum.

Referring to FIG. 2, fraction collector (60), holding sample collection

10 vial (62), is illustrated as capable of sliding movement in one direction. The fraction
collector is a temperature controlled, rack that positions the blood collection vials
underneath the dispensing tip for collection of blood samples. Typically, the fraction
collector will contain a heat exchanger for maintaining a constant vial temperature.
Hie collector also contains a computer controlled motor means for movement of the

15 collector.

Further illustrated in FIG. 2 is a process control scheme (shown by
dotted lines) for connecting the components of the apparatus to computer control
means (58). Digital input/output and fraction collector controller (61) are
electronically connected to each of cannula valve (20), outlet valve (24), and air valve

20 (30). The fraction collector's motor means is interfaced with controller (61) to
provide computer controlled sliding of the collector rack in coordination with the
discharge of samples from the dispensing tip into collection vials. Syringe pump (32),
programmable valve (34) and vacuum means (54) are directly interfaced and
controlled by computer means. The computer means is capable of coordinating and

25 sequencing the opening and closing of the valves, fraction collector, syringe pump,
and programmable valve to facilitate the repetitive, systematic sampling of blood, and
cleaning of the apparatus at desirable times.

In one preferred embodiment of the invention there is described an
apparatus for automatically, repetitively sampling the blood of a plurality of conscious

30 arirmals, each animal being connected to a separate sampling line within a multi-line
.apparatus, characterized as:

a) means for individually confining a plurality of conscious, catheterized
animals;

35

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WO 01/78591 PCT/US01/1 1662

10

b) a plurality of cannulae, each cannula having first and second ends, said first
end connected to the catheterized animal;

c) a plurality of cannula valves, each valve having first and second ends, said
first end connected to the cannula, said cannula valves' opening and closing
being computer controlled;

d) a plurality of cannula T-fittings, each valve having first, second and third ends,
said first end being connected to the second end of each cannula valve;

e) a plurality of outlet valves, each valve having first and second ends, said first
end being connected to the second end of each cannula T-fitting, said outlet
valves' opening and closing being computer controlled;

15 f) a plurality of dispensing tips, each tube having first and second ends, said first
end being connected to the second end of each outlet valve, said second end
being suitable for discharging blood into a sample, collection vial;

g) means for horizontal and vertical movement of the dispensing tips in and out
20 of the collection vial, said means for movement being computer controlled;

h) a plurality of air T-fittings, each valve having first, second and third ends, said
first end being connected to the third end of the cannula T-fitting;

25 i) a plurality of air valves, each valve having first and second ends, said first end
being connected to the second end of each air T-fitting, said air valves*
opening and closing being computer controlled;

j) a plurality of syringe pumps, each pump having first and second ends, said
30 first end being connected to the third end of each air T-fitting, wherein the

pumps are suitable for pumping blood and saline through the apparatus, said
pumps being computer controlled, wherein each series of a) through j)
components comprise a separate sampling channel within the apparatus;

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WO 01/78591

PCT/US01/11662

k) a rotary, multi-port, programmable valve, each port suitable for receiving and
discharging fluids, said first port being connected to the second end of the
syringe pump, optionally the second end of a plurality of syringe pumps
connected to a first end of a common manifold having first and second ends,
5 and the second end of the manifold connected to the first port of the

programmable valve, each port's opening and closing being computer
controlled;

1) a saline solution source connected to the second port of the programmable
10 valve;

a fraction collector, comprising:

i) a base having two horizontally opposed, parallel tracks, and a single track
horizontally located above and perpendicular to the horizontally opposed,
parallel tracks,

ii) a temperature controlled rack in slidable contact with the horizontally
opposed, parallel tracks,

iii) means for sliding the rack along the tracks, said means for sliding the rack
being computer controlled, and

iv) a plurality of sample, collection vials removably arranged in the rack, said
sample collection vials being suitable for receiving blood from the
dispensing tip; and

n) computer control means for programming and coordinating the operation of the
collector, valves and pumps to collect blood samples at desired intervals,

30

wherein the valves, syringe pump, and fraction collector functions are coordinated
and controlled by the computer means to withdraw samples from the animal at
repetitive, programmable intervals and discharge the samples in collection vials.

m)

15
20

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WO 01/78591 PCT/US01/11662

The blood sampling apparatus of the invention also contemplates a
wash and rinse system. The system is characterized as water and acid wash
sources connected to the third and fourth ports, respectively, of the programmable
valve. The acid wash source is useful for cleaning the valves and tubing of residue
5 after several collection cycles have been completed. After an acid wash cycle has
been completed, a water rinse of the valves and tubing is necessary to remove any
residual acid and neutralize the pH of the apparatus. During cleaning operations,
the programmable valve closes the second port, connecting the saline source, and
opens the fourth port, connecting the acid wash source, while the first port remains
10 open. After completing the acid wash cycles to clean the system, one or more

water, rinse cycles are performed to remove any residual acid from the components
of the system. The acid wash and distilled water rinse cycles can be performed.
For a complete cleaning of the apparatus, any animals should be removed.
Typically, after completely rinsing the apparatus, distilled water is inserted into the
15 tubing and valves until the next blood sample is collected.

In FIG. 3, there is illustrated an ak-sakne-blood-saline-air unit-volume
(70) generated within the tubing as representative of the blood sample collected from
the animal during operation of the apparatus. While other benefits might exist, it is
believed that the introduction of air bubbles (at least one air bubble at each end of the
20 unit-volume) acts as a barrier for the additional dilution of the saline-blood-blood
unit-volume with saline. Optionally, the apparatus of the present invention can be
designed to generate an air-blood-air unit-volume if desirable, as will become
apparent to one skilled in the art after reading this disclosure.

Generally, the blood sample is diluted with heparinized saline solution,
25 equal to an amount desired for storage of the sample. Generally, the volume of blood
removed from an animal as blood sample will be replaced within the animal blood
system by an equal volume of saline solution during the sampling process. The three-
phased, blood sample, comprising air, saline solution and blood, is characterized by
air bubbles (72) being exterior to saline solutions (74), and the center of the sample
30 being blood (76). Typically, the saline-blood-saline portion of the sample will be
diffused within one another, i.e. the blood and saline phases will co-mingle. Since
many blood samples are diluted with saline solution to prevent decomposition and
dehydration during storage, a novel process of the invention incorporates the saline
solution into the sample during the collection process. Furthermore, the air bubbles
35 on either side of the sample, unit-volume act as a barrier to prevent further dilution of

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WO 01/78591 PCT/US01/11662

the sample with saline solution during transfer through the tubing and discharge
thereof into the collection vial. In addition, the air bubbles prevent the deposition of
blood, boundary layer on the inside of the tubing.

Generally, referring to HG. 4, there is illustrated several process steps,
5 a) through m), for collecting blood samples for a catheterized animal utilizing the
novel apparatus of the invention. Catheterized animal (12) is placed into a metabolic
cage, and connected to a cannula (16), pre-filled with heparinized saline solution. As
shown in FIG. 4(a), air valve (30) and cannula valve (20) are closed, outlet valve (24)
is opened, multi-port, programmable valve is opened to saline source (36), and

10 syringe pump (32) is activated to push saline solution into the tubing. In accordance
with FIG. 4(b), after outlet valve (24) is closed and cannula valve (20) is opened,
syringe pump (32) is reversed to aspire or draw all the saline solution from cannula
(16) into the tubing, wherein blood is withdrawn from animal (12) into cannula (16).
Referring to FIG. 4(c), cannula valve (20) is closed, outlet valve (24) is opened, and

15 the saline solution withdrawn from cannula (16) is purged from the tubing and
replaced with fresh saline (74) withdrawn from saline source (36). FIG. 4(d)
illustrates the opening of air valve (30), closing outlet valve (24), and aspiring air
bubble (72) into air T-fltting (28). According to HG. 4(e), air valve (30) is closed,
outlet valve (24) opened, and saline solution (74) is use to push air bubble (72)

20 forward towards cannula T-fitting (22) in the tubing. As provided in FIG. 4(f), air
valve (30) is opened, outlet valve (24) is closed, and a second air bubble (72) is
aspired into the tubing at air T-fitting (28). Hie volume of saline solution (74)
between the bubbles being the volume of saline desired to dilute the sample for
storage purposes. In accordance with FIG. 4(g), air valve (30) is closed, outlet valve

25 (24) is opened, and the syringe pump is utilized to push the air bubbles (72) within the
saline solution from the air T-fitting over the cannula T-fitting. After the saline-
portion of the air-saline-air unit-volume is directly over the cannula valve, the cannula
valve is open and the dispensing valve is closed, and with the syringe pump operating
to aspirate or pull saline through the tubing, a desired volume of blood sample is

30 collected into the saline phase of the air-saline-air unit- volume to generate blood
sample (70), as shown in FIG. 4(h). Afterwards, as shown in HG. 4(i), the syringe
pump is deactivated, the cannula valve closed, the dispensing valve opened, and the
pump re-activated to push blood sample (70) towards dispensing tip (26). Referring
to FIG. 4(j), blood sample (70) is discharged from dispensing tip (26) into collection

35 vial (78) for storage and analysis. After blood sample (78) is removed from

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WO 01/78591 PCT/US01/11662

dispensing tip (26), as illustrated in FIG. 4(k), air valve (30) is opened, outlet valve
(24) is closed, and air bubble (72) is generated at air T-fitting (28) by aspiration.
According to FIG. 4(1), air bubble (72) is pushed through the tubing toward cannula
T-fitting (22) until it is a distance therefrom, the distance being equal to the volume of
5 replacement saline necessary to replace the volume of sample removed from the
animal. As shown in FIG. 4(m), outlet valve (24) is closed, cannula valve (20) is
opened, and the syringe pump is activated to push the bubble (72) and replacement
saline into cannula (16). Lastly, referring to FIG. 4(n), the syringe pump continues to
push the replacement saline followed by air bubble (72) down through the cannula

10 until the air bubble is just exterior to the animals body and the blood and saline are
injected into the animal's blood system. When another sample is desired, the air
bubble adjacent to the animal is drawn up the cannula until it reaches the cannula T-
fitting, and the process cycle according to FIG. 4(a) through 4(n) are repeated.

Another embodiment of the invention is directed to a process for

15 automatically, repetitively sampling blood from a conscious animal utilizing the novel
apparatus described herein above, the process characterized by the steps of:

a) closing the cannula and air valves, opening the outlet valve, and the first and
second ports of the programmable valve to the saline source;

20

b) filling the tubing with heparinized, saline solution from the saline source
utilizing the syringe pump to push the solution into the tubing;

c) attaching a catheterized animal to the cannula, wherein the cannula is pre-
25 filled with saline solution;

d) closing the outlet valve, opening the cannula valve, and aspiring the saline
solution from the cannula through the cannula T-fitting into the tubing in a
direction towards the syringe pump until the saline in the cannula is in the

30 tubing and a blood sample from the animal is drawn into the cannula;

e) closing the cannula valve, opening the outlet valve, and purging the tubing,
through the dispensing tip of pre-filled saline solution, while filling the
tubing with fresh, saline solution form the saline source;

35

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WO 01/78591 PCT/US01/11662

f) closing the outlet valve, opening the air valve, and aspiring a first air bubble
into the tubing at the air T-iitting;

g) closing the air valve, opening the outlet valve, and pushing the air bubble
5 towards the cannula T-fitting;

h) closing the outlet valve, opening the air valve, and aspiring a second air
bubble into the tubing followed by closing the air valve, wherein an air-
saline-air phase is formed within the tubing, wherein a volume of saline

10 between the air bubbles is equal to the volume of saline desired to dilute the

sample;

i) closing the air valve, opening the outlet valve, and pushing the air-saline-air
phase through the tubing, towards the cannula T-fitting so that the saline-

15 portion of the phase is over the cannula T-fitting, wherein the tubing is filled

with additional saline solution;

j) opening the cannula valve and activating the syringe pump to introducing an
amount of blood sample into the saline-portion of the air-saline-air phase,
20 wherein an air-saline-blood-saline-air phase is formed in the tubing;

k) closing the cannula valve, and pushing the air-saline-blood-saline-air phase
through the tubing to the dispensing tip;

25 1) discharging the air-saline-blood-saline-air phase through the dispensing tip,

into a vial of the fraction collector, wherein the tubing is filled with
additional saline solution;

m) closing the outlet valve, opening the air valve, and aspiring an air bubble
30 into the tubing at the air T-fitting;

n) closing the air valve, opening the outlet valve, and pushing the air bubble
through the tubing proximal to the cannula T-fitting to form an air-saline
phase adjacent to the cannula T-fitting, wherein a volume of saline in the
35 tubing between the air bubble and the end of the cannula T-fitting connected

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WO 01/78591 PCT/US01/11662

to the cannula valve is equal to the volume of blood withdrawn from the
animal; and

o) closing the outlet valve, opening the cannula valve, said cannula being filled
5 with blood, and pushing the air-saline phase and blood through the cannula

into the animal, via the syringe pump, until the blood and saline are in the
animal and the air bubble is adjacent to the outside of the animal;

wherein during collection of subsequent blood samples, steps a) through o) are
10 repeated, wherein prior to repeating the steps, the air bubble adjacent to the

animal and blood are raised to the cannula T-fitting and purged from the tubing
with saline solution, and wherein the opening and closing of the valves,
operation of the syringe pump and programmable valve are performed via
computer control means.
15 The apparatus and process of the invention are suitable for collection

of blood samples from small animals having a limited blood supply. Generally,
no more than from about 10 to about 20 micro-liters (ul) blood samples are
collected from small animals, e.g., mice, rats, hamsters, etc., per sample. Since
the loss of blood from such small animals could be critical to normal
20 metabolism, the present invention contemplates replacement of the sample with

an equal volume of heparinized, saline solution. Typically, the 10 to 20 ul of
blood sample can be diluted with from about 80 to about 100 ul of heparinized,
saline solution prior to storage or analysis. The samples can be separately stored
and analyzed or composites of an animal can be generated and stored for
25 analyses according to methods known to those skilled in the art.

The means for confining a conscious animal generally include a
metabolic cage or other surroundings, containing sufficient food and water, to
prevent the animal from disconnecting the cannula. In one embodiment of the
invention, a rigid material, metal wire or meshing can be placed around the cannula
30 to prevent the animal from chewing through the cannula. Yet, in another

embodiment of the invention, a fluid-swivel or similar device can be connected
between the cannula and cannula valve to allow complete pivoting of the cannula
as the animal moves about in the confining means. The confining means will
generally comprise a cage with food and water sufficient to maintain the animal's
35 normal metabolism.

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WO 01/78591 PCIYUS01/11662

The cannula and other tubing used to connect the components of the
invention are generally constructed of a flexible polyethylene having an inside
diameter of 0.03 inches and an outside diameter of 0.048 inches. The outlet, air
and cannula pinch valves exhibit an inside diameter of 1/32 inches and an outside
5 diameter of 3/32 inches and are marketed by Bio-Chem Valve Corp, Boston,

Massachusetts. The rigid stainless steel dispensing tip has an inside diameter of
0.023 inches and an outside diameter of 0.033 inches and is marketed by Small
Parts, Miami Lakes, Florida. The syringe pump is marketed by Cairo, Inc,
Sunnyvale, California. The multi-port programmable valve is marketed by Cairo,
10 Inc.

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5

WHAT IS CLAIMED IS:

1 . An apparatus for automatically, repetitively sampling blood
from a conscious animal, comprising:

a) means for confining a conscious, catheterized animal;

b) a cannula having first and second ends, said first end connected to the
catheterized animal, said cannula being suitable for sampling blood from the

10 animal;

c) a cannula valve having first and second ends, said first end connected to the
cannula, said cannula valve opening and closing being computer controlled;

15 d) a cannula T-fitting having first, second and third ends, said first end connected
to the second end of the cannula valve;

e) an outlet valve having first and second ends, said first end connected, via
tubing, to the second end of the cannula T-fitting, said outlet valve opening

20 and closing being computer controlled;

f) a dispensing tip having first and second ends, said first end connected to the
second end of the outlet valve, said second end being suitable for discharging
blood into a sample, collection vial;

25

g) means for horizontal and vertical movement of the dispensing tip, said means
for movement being computer controlled;

h) an air T-fitting having first, second and third ends, said first end connected to
30 the third end of the cannula T-fitting;

i) an air valve having first and second ends, said first end connected to the
second end of the cannula T-fitting, said second end connected to a check
valve;

35

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WO 01/78591 PCT/US01/11662

j) a syringe pump suitable for transferring blood and saline solution through the
apparatus, said pump having first and second ends, said first end connected,
via tubing, to the third end of the air T-fitting, said pump being computer
controlled;

5

k) a rotary, programmable valve having four ports for receiving and discharging
fluids, wherein the ports open and close by computer control, said first port
being connected, via tubing, to the second end of the syringe pump;

10 1) a heparinized saline source comprising a saline solution, said saline source
being connected to the second port of the programmable valve;

m) a fraction collector, comprising:

15 i) a base having two horizontally opposed, parallel tracks, and a single track

horizontally positioned above and perpendicular to the horizontally
opposed, parallel tracks,

ii) a temperature controlled rack in slidable contact with the horizontally
20 opposed, parallel tracks,

iii) means for sliding the rack along the tracks, said means being computer
controlled, and

25 iv) a plurality of sample, collection vials removably located in the rack, said

vials being suitable for receiving samples from the dispensing tip; and

n) computer means for accepting timing commands for collecting samples from
the animal in coordination with the opening and closing of valves, sliding of
30 the temperature controlled rack, pumping of the syringe pump, and dispensing

samples into the collection vials,

wherein samples can be collected at predetermined time intervals.

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WO 01/78591 PCT/US01/1 1662

2. The apparatus according to Claim 1, wherein a water source is
connected to the third port of the multi-port valve.

3. The apparatus according to Claim 2, wherein an acid wash
5 source is connected to the third port of the multi-port valve.

4. The apparatus according to Claim 3, wherein the saline source
comprises a heparinized saline solution.

10 5. The apparatus according to Claim 4, wherein the acid source

comprises from about 0.1 to about 1.0 weight percent of a hydrochloric acid solution,
based on 100 weight percent total.

6 The apparatus according to Claim 5, wherein the computer
15 means further comprises an input/out interface card for operation of the cannula,
outlet and air valves, and fraction collector.

7. The apparatus according to Claim 6, wherein a swivel is
connected between the cannula and the cannula valve.

20

8. An apparatus for automatically, repetitively sampling blood
from a plurality of conscious animals, comprising:

a) means for individually confining a plurality of conscious, catheterized
25 animals;

b) a plurality of cannulae, each cannula having first and second ends, said first
end connected to the catheterized ariimal;

30 c) a plurality of cannula valves, each valve having first and second ends, said
first end being connected to each cannula, said cannula valves* opening and
closing being computer controlled;

d) a plurality of cannula T-fittings, each valve having first, second and third ends,
35 said first end being connected to the second end of each cannula valve;

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WO 01/78591 PCTYUS01/11662

e) a plurality of outlet valves, each valve having first and second ends, said first
end being connected to the second end of each cannula T-fitting, said outlet
valves' opening and closing being computer controlled;

5

f) a plurality of dispensing tips, each tube having first and second ends, said first
end being connected to the second end of each outlet valve, said second end
being suitable for discharging blood into a sample, collection vial;

10 g) means for horizontal and vertical movement of the dispensing tip in and out of
the collection vial, said means for movement being computer controlled;

h) a plurality of air T-fittings, each valve having first, second and third ends, said
first end being connected to the third end of the cannula T-fitting;

15

i) a plurality of air valves, each valve having first and second ends, said first end
being connected to the second end of each air T-fitting, said air Tattings*
opening and closing being computer controlled;

20 j) a plurality of syringe pumps, each pump having first and second ends, said
first end being connected to the third end of each air T-fitting, wherein the
pumps are suitable for pumping blood and saline through the apparatus, said
pumps being computer controlled, wherein each series of a) through j)
components, comprise a separate sampling channel within the apparatus;

25

k) a rotary, multi-port, programmable valve, each port suitable for receiving and
discharging fluids, said first port being connected to the second end of the
syringe pump, optionally the second end of a plurality of syringe pumps
connected to a first end of a common manifold having first and second ends,
30 and the second end of the manifold connected to the first port of the

programmable valve, each port's opening and closing being computer
controlled;

1) a saline solution source connected to the second port of the programmable
35 valve;

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WO 01/78591 PCT/US01/11662

m) a fraction collector, comprising:

i) a base having two horizontally opposed, parallel tracks, and a single track
5 horizontally located above and perpendicular to the horizontally opposed,

parallel tracks,

ii) a temperature controlled rack in slidable contact with the horizontally
opposed, parallel tracks,

10

iii) means for sliding the rack along the tracks, said means for sliding the rack
being computer controlled, and

iv) a plurality of sample, collection vials removably arranged in the rack, said
15 collection vials being suitable for receiving a sample from the dispensing

tip; and

n) computer control means for programming and coordinating the operation of
the collector, valves and pumps to collect blood samples at desired intervals,

20

wherein the valves, syringe pump, and fraction collector functions are coordinated and
controlled by the computer means to withdraw samples from the animal at repetitive,
programmable intervals and discharge the samples in collection vials.

25 9. The apparatus according to Claim 8, wherein a water source is

connected to the third port of the programmable valve.

10. The apparatus according to Claim 9, wherein an acid wash
source is connected to the fourth port of the programmable valve.

30

11. The apparatus according to Claim 10, wherein a swivel is
connected between the cannula and the cannula valve, said swivel being for pivotal
movement of the cannel.

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12. The apparatus according to Claim 1 1 , wherein a waste removal
system is incorporated into the apparatus, said waste removal system comprising a
dispensing tip manifold for receiving the dispensing tips, a waste collection trap
connected to said manifold, a safety trap connected to said waste collection trap, and a

5 vacuum means connected to said safety trap, said vacuum means for removing waste
fluids from the dispensing tip.

13. The apparatus according to Claim 12, wherein the dispensing
tip manifold comprises a base having a plurality of vertical holes therethrough, each

10 hole for receiving a dispensing tip, and a single hole horizontal hole intersecting with
each vertical hole, said vertical hole for the passage of air associated with the vacuum
means.

14. The apparatus according to Claim 13, wherein the cannula,

1 5 outlet and air valves are selected from the group consisting of pinch and check valves.

15. The apparatus according to Claim 14, wherein cannula, outlet
and air valves are interfaced with an input/output controller prior to connecting to the
computer means.

20

16. The apparatus according to Claim 15, wherein the slidable,
fraction collector is interfaced with an input/output controller prior to connecting to
the computer means.

25 17. The apparatus according to Claim 16, wherein the saline source

comprises a heparinized saline solution.

18. The apparatus according to Claim 17, wherein a water source is
connected to the third port of the programmable valve.

30

19. The apparatus according to Claim 18, wherein an acid wash
source is connected to the fourth port of the programmable valve.

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20. The apparatus according to Claim 19, wherein the acid wash
source comprises from about 0.1 to about 1.0 weight percent of a hydrochloric acid
solution, based on 100 weight percent total.

5 21 . The apparatus according to Claim 20, wherein an air-saline-

blood-saline-air phase is generated in the apparatus.

22. The apparatus according to Claim 21, wherein the air-saline-
blood-saline-air phase is the blood sample.

10

23. The apparatus according to Claim 22, wherein the saline-blood-
saline portion of the sample is diffused.

24. A process for automatically, repetitively sampling blood of a
15 conscious animal, utilizing the apparatus according to Claim 1, comprising the steps

of:

a) closing the cannula and air valves, opening the outlet valve, and the first and
second ports of the programmable valve to the saline source;

20 b) filling the tubing with heparinized, saline solution from the saline source

utilizing the syringe pump to push the solution into the tubing;

25

30

c) attaching a catheterized animal to the cannula, wherein the cannula is pre-
filled with saline solution;

d) closing the outlet valve, opening the cannula valve and aspiring the saline
solution from the cannula through the cannula T-fitting into the tubing in a
direction towards the syringe pump until the saline in the cannula is in the
tubing and a blood sample form the animal is drawn into the cannula;

e) closing the cannula valve, opening the outlet valve, and purging the tubing,
through the dispensing tip of pre-fLUed saline solution, while filling the
tubing with fresh, saline solution form the saline source;

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WO 01/78591

PCIYUS01/11662

f) closing the outlet valve, opening the air valve, and aspiring a first air bubble
into the tubing at the air T-fitting;

g) closing the air valve, opening the outlet valve, and pushing the air bubble
5 through the tubing towards the cannula T-fitting;

h) closing the outlet valve, opening the air valve, and aspiring a second air
bubble into the tubing followed by closing the air valve, wherein an air-
saline-air phase is formed within the tubing, wherein the volume of saline

10 between the air bubbles is equal to the volume of saline desired to dilute the

sample;

i) closing the air valve, opening the outlet valve, and pushing the air-saline-air
phase through the tubing, towards the cannula T-fitting so that the saline-

15 portion of the phase is over the cannula T-fitting, wherein the tubing is filled

with additional saline solution;

j) opening the cannula valve and activating the syringe pump to introducing an
amount of blood sample into the saline-portion of the air-saline-air phase,
20 wherein an air-saline-blood-saline-air phase is formed in the tubing;

k) closing the cannula valve, and pushing the air-saline-blood-saline-air phase
through the tubing to the dispensing tip;

25 1) discharging the air-saline-blood-saline-air phase through the dispensing tip,

into a vial of the fraction collector, wherein the tubing is filled with
additional saline solution;

m) closing the outlet valve, opening the air valve, and aspiring an air bubble
30 into the tubing at the air T-fitting;

n) closing the air valve, opening the outlet valve, and pushing the air bubble
through the tubing proximal to the cannula T-fitting to form an air-saline
phase adjacent to the cannula T-fitting, wherein a volume of saline in the
35 tubing between the air bubble and the end of the cannula T-fitting connected

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WO 01/78591 PCT/US01/11662

to the cannula valve is equal to the volume of blood withdrawn from the
animal; and

o) closing the outlet valve, opening the cannula valve, said cannula being filled
5 with blood, and pushing the air-saline phase and blood through the cannula

into the animal, via the syringe pump, until the blood and saline are in the
animal and the air bubble is adjacent to the outside of the animal;

wherein during collection of subsequent blood samples, steps a) through o) are
repeated, wherein prior to repeating the steps, the air bubble adjacent to the animal
and blood are raised to the cannula T-fitting and purged from the tubing with saline
solution, and wherein the opening and closing of the valves, operation of the
syringe pump and programmable valve are performed via computer control means.

25. The process according to Claim 24, wherein the blood sample
comprises an air-saline-blood-saline-air phase.

26. The apparatus according to Claim 25, wherein the saline-blood-
saline portion of the sample is diffused.

20

10

15

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10/10

INTERNATIONAL SEARCH REPORT

International application No.
PCT/USO 1/11662

A. CLASSIFICATION OF SUBJECT MATTER

IPC<7) : A61B 5/00; B65D 81/00

USCL : 600/573
According to Interna timwl pate nt Classification (IPO or to both national classification and IPC

FIELDS SEAR

Mmimum docamentatiQn searched (classification system followed by classification symbols)
U.S. : 600/573, 576, 577, 578, 579; 604/253, 27

Documentation searched other than miniTrmm documentation to the extent thai such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
WEST: automatic, automated, blood collection, blood sample, catheter, computer, saline, valve, T- fitting, intervals

DOCUMENTS CONSIDERED TO BE RELEVANT

Category

Citation of document, with mdication, where appropriate, of the relevant passages

Relevant to claim No.

A
A
A

US 4,509,534 A (TAS5IN, JR.) 09 April 1985, see whole document.
US 4,722,725 A (SAWYER et aJ) 02 February 1988, see whole document.
US 5,417,673 A (GORDON) 23 May 1995, see whole (locumenL

1-26
1-26
1-26

□

Further documents are listed in the continuation of Box C.

□

See patent family annex.

* Special categories of cited dactnnenls!

"A* document drihing the gcncntf stale of the art which b not considered to
be of particular relevance

"B" earlier ippHeatlm nr patent pnafahed op eg after tha tntftmtj^il filing
date

"L" document which may throw doubts no priority dalm(i) or which U cited
to establish the publication dale of another citation or other special res on
(at ipecifled)

"O* document referring to an oral dtsdontre, use, exhibition or other means

"P* itnmmw|t pihi^h^^ prl"f immnrim^ fni^g dm* frril htrt thm thf
-lutadta^datexbliaed

"T" later dfyp"!* pobUihed after the International filing date or

priority date and not In conflict with the application but cited to
understand the principle or theory underlying the tnvc&tioa

"X* document of parUcnlar relevance; (he claimed invention cannot be

considered novel or cannot be considered to Involve an inventive
step when the document b taken alone

*Y" dosnmrnt of particular relevance; the claimed invention cannot be

considered to involve an Inventive step vhea the document b
combined with one or more other roca docnmenti, such
combination being obvious to a peace skilled In the art

"ft* doenmrnt merober of the same patent family

Date of the actual completion of the international search
16 July 2001 (16.07.2001)

Name and rpwj lfaj * address of die ISA/US

Ccanmtsstoner of Patents and Trad ema rks
BcotPCT

Washington. D.C. 20231
Facsimile No. (703)305-3230

lg of the international search report

Brian Szmal

Telephone No. (703)308-0858

Form PCT/ISA/210 (second sheet) (July 1998)